A novel inhibitor of the mitochondrial respiratory complex I with uncoupling properties exerts potent antitumor activity

Cancer cells are highly dependent on bioenergetic processes to support their growth and survival. Disruption of metabolic pathways, particularly by targeting the mitochondrial electron transport chain complexes (ETC-I to V) has become an attractive therapeutic strategy. As a result, the search for clinically effective new respiratory chain inhibitors with minimized adverse effects is a major goal. Here, we characterize a new OXPHOS inhibitor compound called MS-L6, which behaves as an inhibitor of ETC-I, combining inhibition of NADH oxidation and uncoupling effect. MS-L6 is effective on both intact and sub-mitochondrial particles, indicating that its efficacy does not depend on its accumulation within the mitochondria. MS-L6 reduces ATP synthesis and induces a metabolic shift with increased glucose consumption and lactate production in cancer cell lines. MS-L6 either dose-dependently inhibits cell proliferation or induces cell death in a variety of cancer cell lines, including B-cell and T-cell lymphomas as well as pediatric sarcoma. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI-1) partially restores the viability of B-lymphoma cells treated with MS-L6, demonstrating that the inhibition of NADH oxidation is functionally linked to its cytotoxic effect. Furthermore, MS-L6 administration induces robust inhibition of lymphoma tumor growth in two murine xenograft models without toxicity. Thus, our data present MS-L6 as an inhibitor of OXPHOS, with a dual mechanism of action on the respiratory chain and with potent antitumor properties in preclinical models, positioning it as the pioneering member of a promising drug class to be evaluated for cancer therapy. MS-L6 exerts dual mitochondrial effects: ETC-I inhibition and uncoupling of OXPHOS. In cancer cells, MS-L6 inhibited ETC-I at least 5 times more than in isolated rat hepatocytes. These mitochondrial effects lead to energy collapse in cancer cells, resulting in proliferation arrest and cell death. In contrast, hepatocytes which completely and rapidly inactivated this molecule, restored their energy status and survived exposure to MS-L6 without apparent toxicity.

This figure reports the main chemical steps of OXPHOS, including the four protein complexes of the electron transport chain (ETC) and ATP synthase, with classical substrates and inhibitors used in the experiments.Briefly, the respiratory chain can be fed by electrons coming from NADH (equivalent to Glutamate/Malate), succinate or TMPD/Ascorbate (artificial electron donor) through ETC-I, ETC-II and ETC-IV, respectively.These electrons are then transferred to ETC-IV where they are accepted by O 2 , the ultimate electron acceptor.The forward electron flux through the ETC drives proton pumping, resulting in a proton gradient across the IMM.Finally, ATP synthase exploits the energy of this gradient to drive the phosphorylation of ADP into ATP.The chemical structures of ETC-1 inhibitors used in these study is indicated in figure S1B

S2A: Interaction/competition between MS-L6 and decylubiquinone.
Left panel shows ETC-I activity measured by following NADH absorbance spectrophotometric assay at 340nM of rat liver sub-mitochondrial particles in the presence of DMSO (vehicle control) or 50µM MS-L6.Sub-mitochondrial particles were first incubated with 1 mM NADH in the presence of 1mM KCN, and then increasing concentrations of decylubiquinone were added.Decylubiquinone did not inhibit ETC-I activity under control condition but higher concentrations of decylubiquinone tended to inhibit NADH oxidation, especially in the presence of 50 µM MS-L6(Fig.S2A).However, the relief of MS-L6 inhibition of NADH oxidation was observed at 50 µM MS-L6 in the presence of 20 µM decylubiquinone (Fig. S2B) S2B: BSA has no effect rotenone activity.
Left panel shows ΔΨm of 0.5mg/mL of intact rat liver mitochondria energized with ETC-I substrate (Glut/Mal), after treatment with 1µM rotenone in the presence (grey lane) or absence (blue lane) of 0.15% BSA.When indicated 800 nM FCCP was added to fully depolarize ΔΨm.The depolarization was slightly lower in the presence of BSA, presumably due to chelation of free fatty acids.
Right panel shows the OCR of submitochondrial particles energized with NADH in the presence or absence of 0.15% BSA.The decrease in OCR induced by rotenone was similar in both conditions.

S2C: Quantification of the MS-L6 uncoupling effect.
Left panel shows ΔΨm of 0.5mg/mL of intact rat liver mitochondria energized with ETC-II substrate (succinate), after treatment with 50 µM MS-L6 (blue lane) or increasing concentrations of FCCP (green lane).Control (Brown trace).MS-L6-induced depolarization was between that induced by 50nM and 100 nM FCCP.
Right panel shows oxygen consumption of mitochondria with succinate in the presence or absence of 50 µM MS-L6 or 100 nM FCCP.Where indicated 400µM ADP was added.Oxygen consumption during ATP synthesis was higher in the presence of 50 µM MS-L6 or 100 nM FCCP than in the control condition.
S2D: At the same uncoupler concentration, the depolarizing effect depends on the Vmax of the respiratory chain.Panels show ΔΨm of 0.5mg/mL of intact rat liver mitochondria energized with ETC-I substrate (Glut/Mal) or ETC-II substrate (Succ).ETC-I was then inhibited with either rotenone (left) or MS-L6 (right) followed by sequential addition of FCCP.In the presence of rotenone (left), 50 nM FCCP completely depolarized mitochondria fed with ETC-I substrate, whereas it induced a visible but weak depolarization when mitochondria were fed with ETC-II substrate (succinate).When 50 µM MS-L6 was added instead of rotenone, it completely depolarized mitochondria fed with ETC-I substrate, whereas it induced a visible but weak depolarization when mitochondria were fed ETC-II substrate (succinate).Right panel shows histograms of quantification of these analyses, presented as the percentage of cells in the different apoptosis stages of all well replicates.Data are presented as mean ± SD, and from one representative experiment n>3.
Statistical significance was determined as described in materials and methods.Statistical significance was determined as described in materials and methods.

Effect of rotenone on cells overexpressing NDI1
For OCR measurement of cells overexpressing or not NDI1 frozen cells (10 million cells) were thawed and placed directly into ultrapure water supplemented with 10 mM Pi for 5 min (osmotic shock).The respiratory chamber was then sealed with a cap and 0.5 mM NADH, 1 µM rotenone, 5 mM succinate and 100µM antimycin A were added sequentially.

Effect of BSA on rotenone sensitivity
ΔΨm of rat liver mitochondria incubated in the presence of ECT-I substrate was evaluated with the mitochondrial probe, rhodamine 123 as described above.Rotenone (1µM) was added in the presence or absence of BSA (0.15%).
The effect of rotenone (1µM) was also measured by OCR in sub-mitochondrial particles (1 mg/ml) in the presence or absence of BSA (0.15%).

Quantification of the MS-L6 uncoupling effect.
ΔΨm of rat liver mitochondria incubated in the presence of ECT-II substrate was evaluated with the mitochondrial probe, rhodamine 123 as described above.Increasing concentrations of FFCP were used to progressively abolish ΔΨm and were compared to the depolarization induced by 50 µM MS-L6.

Effect of FCCP on ΔΨm when mitochondria were incubated with rotenone in the presence of ECT-I or ECT-II substrate, respectively.
ΔΨm of rat liver mitochondria incubated in the presence of ECT-I or ECT-II substrates was evaluated with the mitochondrial probe, rhodamine 123 as described above.Rotenone (1 µM) was then added and increasing concentrations of FFCP were used to progressively abolish ΔΨm.

Measurement of OCR and ECAR in intact cells using Seahorse technology:
Real-time metabolic analysis was performed using the Seahorse Bioscience XFe96 Extracellular Flux Analyzer (Agilent), which allows simultaneous measurement of cellular oxygen consumption rate (OCR in pmol/min) and extracellular acidification rate (ECAR in mpH/min).On the day of the assay, 1.5 x 10 5 RL and 2 x 10 5 K422 cells in exponential growth were seeded onto Seahorse 96-well plates coated with Corning Cell-Tak (TMsub), according to the manufacturer's instructions.The number of cells seeded was optimized to ensure 70-80% confluence.The culture medium was replaced with Seahorse XF RPMI assay medium pH 7.4 (Agilent) and the plate was pre-incubated for 30 min at 37°C in a non-CO2 incubator.
Simultaneous OCR and ECAR were then measured according to supplier's instructions under basal conditions (injection of inhibitor diluent i.e., DMSO) and after sequential injections of different inhibitors (MS-L6, rotenone or IACS-010759).OCR and ECAR levels were normalized to the number of cells per well using Agilent Seahorse XF imaging and cell counting procedures.

Live/dead cell analysis
The day before treatments, cells were seeded in 80µL/well of RPMI (1% penicillin-

Table of immune populations analyzed
Viable immune populations identified (Annexin negative and CD45 positive) CD45 is used to assess possible non-leukocyte contamination of the lymphocyte window in the graphical analysis.

Figure S1 :
Figure S1: Schematic diagram of mitochondrial respiratory chain and inhibitors.This figure reports the main chemical steps of OXPHOS, including the four protein complexes of the electron transport chain (ETC) and ATP synthase, with classical substrates and inhibitors used in the experiments.Briefly, the respiratory chain can be fed by electrons coming from NADH (equivalent to Glutamate/Malate), succinate or TMPD/Ascorbate (artificial electron donor) through ETC-I, ETC-II and ETC-IV, respectively.These electrons are then transferred to ETC-IV where they are accepted by O 2 , the ultimate electron acceptor.The forward electron flux through the ETC drives proton pumping, resulting in a proton gradient across the IMM.Finally, ATP synthase exploits the energy of this gradient to drive the phosphorylation of ADP into ATP.The chemical structures of ETC-1 inhibitors used in these study is indicated in figureS1B Figure S1

Figure S5 :
Figure S5: Analysis of MS-L6 effects on human PBMCs by flow
streptomycin, 10% SVF) in a 96-well plate with a transparent bottom.Treatments were added the next day in 20µL/well at the appropriate dilution to obtain the final working concentration of each compound.After 48h of treatment, the simultaneous determination of live and dead cells by imaging was performed using the LIVE/DEAD Viability/Cytotoxicity Assay Kit (L3224 Invitrogen) according to manufacturer's protocol.This two-color fluorescence cell viability assay is based on the simultaneous detection of live and dead cells with two probes subcutaneously into SCID mice (females, 4 weeks).To circumvent the heterogeneity of tumor growth, 2 x 2mM pieces of a first tumor were then surgically implanted into the flanks of other animals.This set of mice was used for the efficacy study.Mice were randomized when tumors reached a mean volume of 100mM 3 for the 2 groups (control and L6).All mice were observed to detect any toxic effects of the product.The endpoints are defined by animal ethics as tumor diameter of > 18mm, significant weight loss or changes in animal well-being.To assess the efficacy of the compounds on tumorigenesis, tumor volume was measured three times per week.Primary tumor sizes were measured with calipers and tumor volume (TV) was extrapolated to a sphere using the formula TV= 4/3 π x r3, by calculating mean radius from the two measurements.The median and standard deviation were also calculated for each group.Median is preferred to mean to exclude extreme values.MS-L6 was administered by intraperitoneal injection five times per week, at a dose of 50mg/kg.Control DMSO was also administered by intraperitoneal injection five times per week.